Different Corneal Epithelial Healing Mechanisms
in Rat and Rabbit: Role of Actin and Calmodulin
H. Kaz Soong and Charles Cintron
The authors investigated the effects of calmodulin inhibitors, trifluoperazine (10-20 juM) and W-7
(25-50 juM), and of cytochalasin B (5 ^g/ml) on the F-actin distribution, surface morphology, and
migration of rat and rabbit corneal epithelial cells in tissue culture. In the rat, actively migrating cells
have abundant F-actin-containihg stress fibers and numerous cytoplasmic extensions of the plasmalemma. These features, and ultimately cell migration, are inhibited by calmodulin inhibitors and
cytochalasin B. In the rabbit, migrating cells are devoid of stress fibers and cytoplasmic extensions.
Cell migration is not inhibited by calmodulin inhibitors but is arrested by cytochalasin B. The cellto-substrate adhesion is reduced by calmodulin inhibitors in both rat and rabbit. These findings
corroborate our earlier observations in organ culture studies and support the view that corneal
epithelial cell migration is calmodulin-dependent in the rat, while it is not in the rabbit. The complete
blockage of migration in both species by cytochalasin B suggests that actin polymerization is critical
for corneal epithelial locomotion in both species. Invest Ophthalmol Vis Sci 26:838-848, 1985
regulatory protein that mediates many Ca2+-related
cellular events.5 The involvement of CaM in the
control of cell motility in nonmuscle cells is well
documented. 6 Also, CaM is intimately associated
with actin-binding proteins7"9 and has been identified
as a regulator of myosin light chain kinase and Ca 2+ activated myosin ATPase activities.610"12 The effects
of CaM inhibitors, trifluoperazine (TFP) and N-6aminohexyl-5-chloro-l -naphthalene sulfonamide (W7), on corneal epithelial migration in rat and rabbit
have been studied in wounded organ-cultured corneas.13 In that study, TFP and W-7 was found to
inhibit migration in rat in a dose-dependent manner
but had no effect in rabbit. Also, cytochalasin B (CB)
was found to completely inhibit corneal epithelial
cell migration in both species.413
We report here striking differences between the
healing corneal epithelium in rat vs rabbit, vis-a-vis
the filamentary actin (F-actin) distribution and the
alterations of this distribution in response to CaM
inhibitors in cell culture. These findings corroborate
our previous wound-healing studies in organ culture
and demonstrate that the corneal epithelial movement
in rat is CaM-dependent, while it is not in rabbit.
Actin and its role in nonmuscle cell motility is
well documented. 1 ' 2 Its presence, distribution, and
role in epithelial movement during wound healing
have been studied with myosin subfragment-1 labeling
in the rat corneal epithelium by. Gipson and Anderson.3 They observed that actin filaments in normal
rat corneal epithelium form an apical network under
microplicae of superficial cell layers. In contrast, actin
filaments in migrating epithelium were concentrated
in basal regions of cells. Here they were arranged in
parallel bundles of filaments extending back into the
cell from the leading edge and as dense networks at
leading edges and within cellular processes. Gipson
et al also reported that corneal epithelial movement
in rat is inhibited by cytochalasins B and D, both
antagonists of actin polymerization.4
Calmodulin (CaM) found in all eukaryotic cells so
far examined, is a multifunctional, calcium-binding
From the Eye Research Institute of Retina Foundation and the
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary
(Harvard Medical School), Boston, Massachusetts.
Presented in part at the Annual Meeting of the Association for
Research in Vision and Ophthalmology, Sarasota, Florida, April
30-May 4, 1984.
Supported in part by grants EY-05649 and EY-01199 from the
National Institutes of Health, and grants-in-aid from Fight for
Sight, Inc. (New York, NY) and the National Society to Prevent
Blindness.
Submitted for publication: September 25, 1984.
Reprint requests: H. Kaz Soong, MD, Assistant Professor, WK
Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann
Arbor, MI 48105.
Materials and Methods
Cells
Adult male Sprague-Dawley rats and albino rabbits
were treated in accordance with the ARVO Resolution
on the Use of Animals in Research. Full-thickness
808
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ACTIN AND CALMODULIN IN CORNEAL EPITHELIAL HEALING / Soong ond Gnrron
sheets of pure rat and rabbit corneal epithelium were
removed using Dispase II.14 The sheets were allowed
to adhere to the substratum on tissue culture slides
(Miles Laboratories; Naperville, IL) and the cells were
allowed to migrate for 2 to 3 days. Confluency was
avoided. Cells were cultured at 37°C and 5% CO 2 in
Eagle's minimum essential medium (M. A. Bioproducts; Walkersville, MD) with nonessential amino
acids, L-glutamine, penicillin, streptomycin, amphotericin B, and 10% fetal calf serum. Cells were grown
on both glass and collagen (Vitrogen 100, Flow
Laboratories; McLean, VA) in order to assure that
our results were not influenced by peculiarities in one
type of substratum. The cells were initially cultured
in drug-free medium for 2 to 3 days until there was
sufficient spreading and migration onto the substratum. For the drug studies, this medium was then
replaced with medium containing TFP, W-7, or CB,
and the cells were cultured for an additional 24 hr
before examination.
Drugs
TFP (a gift of Smith, Kline, & French; Philadelphia,
PA) or W-7 (CAABCO, Inc.; Houston, TX) was
dissolved in the cell culture medium and protected
from light. Drug-free medium served as the control
for TFP and W-5, an inactive analogue of W-7, was
used as a control for W-7. CB (Sigma Chemical; St.
Louis, MO) was solubilized in 0.1% dimethylsulfoxide
(DMSO) before being added to the medium. DMSO
at this concentration has been found to have no effect
on corneal epithelial migration and ultrastructure.4
DMSO (0.1%) alone, dissolved in drug-free medium,
served as the control for CB.
Fluorescence and Scanning Electron Microscopy
Cultured cells on slides were washed in phosphatebuffered saline (PBS), pH 7.0, fixed in 3.7% formaldehyde for 10 min at 20°C, washed again in PBS,
and extracted with acetone at —20°C for 4 min. The
cells were then immersed in the dark for 40 min in
NBD-phallacidin (Molecular Probes; Junction City,
OR), the F-actin binding fluorescent derivative of the
mushroom phallotoxin, used at a dilution of 165 ng/
ml. As a control for the specificity of the staining,
the cells were preincubated with excess nonfluorescent
phalloidin (Sigma Chemical; St. Louis, MO) (3.3 mg/
ml) to saturate the phallotoxin-binding sites. The
cells were then post-treated with fluorescent phallotoxin. A faint autofluorescence was seen in the cells
of the control specimens (Fig. 1). Fluorescence and
phase-contrast microscopy were carried out in a Zeiss
photomicroscope III.
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Fig. 1. Control for specificity of fluorescent staining by NBDPh. A, Phase micrograph. B, Epifluorescence microscopy of cells
in A. Minimal tissue autofluorescence is present (bar = 10 fim).
Specimens for scanning electron microscopy were
fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate
buffer, pH 7.2, rinsed in 0.2 M sodium cacodylate
buffer following fixation, dehydrated with a graded
series of alcohols, and critical-point dried in CO 2 .
The samples were sputter coated with gold and viewed
with an AMR-1000 scanning electron microscope.
Measurement of Epithelial Migration in
Cell Culture
The length of the path of cell migration in 48 hr
was measured in cell cultures of corneal epithelium
of rat and rabbit, on both glass and collagen substrata.
For these experiments, cells were cultured on special
tissue culture dishes with grid markings of 2-mm
squares on the bottom (Nunc C ; Roskilde, Denmark).
Ten rat and 10 rabbit cell cultures were used for this
purpose. Half of the cultures from each species were
grown on glass and half were grown on collagen
substrata. At 24 hr after explantation, upon sufficient
attachment of the cells to the substratum, the position
of the leading edge relative to the grid markings was
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / June 1985
Vol. 26
Fig. 2. Cells at the leading edge of
migrating epithelium in rat. A, Phase micrograph. B, Fluorescent staining for Factin shows rich arrays of actin-containing
stress fibers, which occasionally converge
into fluorescence dense focal spots (bar
= 30 nm).
determined at four points (spaced 500 ^m apart).
These readings were further facilitated with a micrometer reticle on the ocular eyepiece of the microscope (magnifications of XI00 and X35O). Fortyeight hours after starting the measurements, the linear
displacements of the four predetermined points were
measured. In performing these measurements, several
assumptions were made: (1) the direction of cell
movement is perpendicular to the leading edge, and
(2) all parts of the leading edge move at the same
rate. Measurements of rate were averaged separately
by species and substratum type. Statistical analysis
was done using the nonparametric Mann-Whitney
U-test.
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Results
Cell Morphology, F-Actin Distribution, and
Migration in the Controls
The drug-free controls of rat and rabbit corneal
epithelium in cell culture migrated well on either
glass or collagen substrata but displayed prominent
differences. In rat, the leading edge of the migrating
epithelial cells was lined with cells possessing abundant
lamellopodia, filopodia, and ruffled membranes, as
characteristic of motile cells. Cells in this region were
richly endowed with networks of parallel and crisscrossing arrays of actin-containing stress fibers existing
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ACTIN AND CALMODULIN IN CORNEAL EPITHELIAL HEALING / 5oong ond Gnrron
V
Fig. 3. Actin staining within a 1amellopodium in an epithelial leading
edge cell of rat. Numerous stress fibers
terminate in fluorescence-dense ends
on the cell membrane (arrowheads)
and in intracytoplasmic focal junctions
(asterisks). Ruffled membranes (large
arrows) are visible (bar = 2 ^m).
in multiple focal planes (Fig. 2). Stress fibers often
terminated in fluorescence-dense ends on the plasmalemma (Figs. 3, 4) corresponding to membraneassociated adhesion plaques (see Discussion).15"18 Such
terminations were especially numerous within lamellopodia and ruffled membranes where stress fibers
were abundant. Filopodia stained intensely for Factin. Within the cytoplasm, stress fibers converged
to fluorescence-dense focal points (Fig. 3) corresponding to cytoplasmic dense bodies.15"18 The intracytoplasmic localization of these focal points was confirmed by focusing the microscope up and down. The
central region, well behind the leading edge, was at
least 4 to 5 cell layers thick, in contrast to the leading
edge, which was usually tapered to a single cell layer
thickness. The cells in the central region possessed
fewer stress fibers (Fig. 5) than their counterparts at
the leading edge and there was an increased concentration of F-actin in the cell periphery. The F-actin
distribution in cultured rat corneal epithelial cells,
especially with respect to stress fiber formation and
distribution, was similar to that seen in other cell
types.16'19"23
In the drug-free controls of rabbit, the migrating
corneal epithelium in cell culture appeared markedly
different from rat. In rabbit, the cells at the leading
Fig. 4. Actin distribution at leading
epithelial edge in rat. Note abundance
of stress fibers, some with membraneassociated terminals (large arrow). Filopodia containing actin stress fibers
(small arrows) and ruffled membranes
(curved arrows) are present (bar = 10
Mm).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1985
Fig. 5. Actin distribution in corneal epithelial cells (rat) in the
central region away from the leading edge. Stress fibers here arc
fewer than at the leading edge. F-actin is predominantly concentrated
in the cortical cytoplasm, thereby delineating the cell borders (bar
= 10 (im).
edge were devoid of lamellopodia, filopodia, and
ruffled membranes (Figs. 6, 7). F-actin-containing
stress fibers were extremely rare, if not absent, in all
cells, both at the leading edge and away from the
leading edge. F-actin was mainly concentrated in the
cortical cytoplasm in the periphery of the cells (Figs.
6, 7). The leading edge of the epithelum was one cell
layer thick, while the region away from the leading
edge was about 5 to 6 cell layers thick.
The mean rates of linear migration of the leading
epithelial edge in tissue culture (drug-free controls)
are shown in Table 1. There were no major differences
in migration rates between rat and rabbit on glass (P
> 0.20) or on collagen {P > 0.50) substrata. Furthermore, migration rates of both species were significantly
faster on collagen than on glass (P < 0.0005).
Effects of CaM Inhibitors and CB on Cell
Migration and F-actin
CaM inhibitors had a profound effect on cultured
rat corneal epithelium but had no effect in rabbit. In
rat, incubation of the migrating cells with TFP (1020 ^M) or W-7 (25-50 /zM) was associated with
changes in F-actin distribution, cell shape, and migration. The morphologic alterations were more pronounced at the leading edge than in the region behind
it; but, nevertheless, stress fibers were markedly reduced everywhere, leaving only a diffuse residual
cytoplasmic fluorescence (Fig. 8). In the majority of
cells, F-actin was now concentrated in the cortical
cytoplasm, thus somewhat resembling the appearance
of the drug-free controls of rabbit. Lamellopodia,
filopodia, and ruffled membranes were markedly re-
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Vol. 26
duced in numbers. Scanning electron micrographs of
CaM inhibitor-treated epithelium showed a total loss
of microplicae in the leading edge cells, with the cell
surfaces abnormally smooth in comparison to the
drug-free controls (Figs. 9 A, B). Filopodia, ruffled
membranes, and other cell membrane extensions
were conspicuously absent. Advancing edges of cells
in this area were often curled off the substratum
because of reduced cell-to-substratum adhesion. In
contrast, the cell surface morphology in the regions
away from the leading edge appeared relatively unaffected by CaM inhibitors, as illustrated by intact
microvilli and microplicae (Fig. 9C). Furthermore,
epithelial migration was completely inhibited by TFP
and W-7 in rat.
When these cells were washed free of the drug,
they restored their original shapes; reformed arrays
of stress fibers; reextended lamellopodia, filopodia,
and ruffled membranes; and resumed migration (micrographs not shown). Concentrations of TFP > 40
MM and W-7 > 50 /JM irreversibly inhibited stress
fibers and migration.
In contrast to rat, TFP (10-20 /*M) or W-7 (2550 fiM) in rabbit produced no discernible changes in
actin distribution, cell shape, and migration (micrographs not shown). At higher doses of CaM inhibitors,
however, cell migration was irreversibly arrested.
Moreover, the cells were often detached as a sheet
from the substratum. These irreversible changes are
similar to those seen at higher drug concentrations
in rat and corroborate the toxic reactions seen in
organ cultured corneas at the higher doses of CaM
inhibitors used.13
CB (5.0 Mg/ml) completely, but reversibly, arrested
migration of both rat and rabbit cells in culture. In
rat, CB caused loss of stress fibers. In both rat and
rabbit, CB caused cell rounding (Fig. 10). In rat,
multiple fluorescent spots were diffusely scattered
throughout the cytoplasm, while in rabbit, similar
fluorescent spots delineated the cell membranes. These
spots correspond to focal areas of greatest cell-tosubstratum adhesion.24
Discussion
Actin is a major constituent of eukaryotic cells,
accounting for about 10% of the total proteins in
nonmuscle cells.25 Fluorescent antibody techniques
have revealed actin, myosin, alpha-actinin, and other
accessory contractile proteins within stress fibers.26
Stress fibers have been observed in vitro and in situ,26
and have been strongly implicated in cell migration,25-27 cytoskeletal shape changes,28 and adhesion
to substrata.2529 Although actin is universally involved
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ACTIN AND CALMODULIN IN COP-NEAL EPITHELIAL HEALING / Soong and Cinrrdn
Fig. 6. Cells at the leading edge
of migrating epithelium in rabbit.
A, Phase micrograph. B, Fluorescent
staining for F-actin is concentrated
in the cortical cytoplasm of cells
and reveals a dearth of stress fibers.
Cells are round or polygonal and
are packed uniformly together (bar
- 30 nm).
in eukaryotic cell motility, there is evidence that
stress fibers, per se, are not.30
Our study compares the migration of cultured
corneal epithelial cells from two mammalian species,
specifically with respect to F-actin localization, stress
fiber formation, and regulation by CaM. In rat,
migrating cells were characterized by numerous plasmalemma extensions and rich arrays of stress fibers,
especially in the leading edge cells. According to
DiPasquale,31 the epithelium in a culture is attached
to substatum only at the leading edge, where the
outward movement of the leading cells generates
tensile forces for epithelial spreading. Whether tensile
forces are generated by the stress fibers themselves or
whether they are formed passively in response to
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exogenous forces remains unclear. Studies have shown
these structures to generate contractile forces in vitro.32
In our study, focally fluorescent stress fiber endings
corresponding to adhesion plaques (dense plaques)
and cytoplasmic dense bodies were abundant in rat.
Adhesion plaques were most numerous within cytoplasmic extensions and are associated with focal
zones of stress fiber-cell membrane interactions. These
interactions may govern movement of the cell membrane and adhesion of the cell to the substratum.15
Stress fibers also insert into cytoplasmic dense bodies.15-33 These focal junctions may possibly serve as
central anchoring points whose function is to organize
and distribute tensile forces.
In rabbit, migrating cells were devoid of cytoplasmic
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1985
Vol. 26
Fig. 7. Actin distribution of corneal epithelial cells (rabbit) in
the central region away from the leading edge. F-actin is concentrated
in the cortical cytoplasm of cells. Stress fibers are absent (bar = 10
nm).
extensions, stress fibers, adhesion plaques, and dense
bodies. Despite these differences, cells from both
species migrated equally well on a given substratum.
As a rule, migration was significantly faster on collagen
than on glass.
Since stress fibers disappear upon treatment with
CaM inhibitors in the rat, the formation of stress
fibers appears to be either directly or indirectly controlled by CaM. Moreover, the disappearance of these
structures is accompanied by the loss of cytoplasmic
extensions and ultimately, the complete cessation of
migration. In contrast, in the migrating rabbit epithelium, normally devoid of stress fibers and cytoplasmic
extensions, CaM inhibitors did not alter the existing
F-actin distribution or the cell surface morphology;
most remarkably, however, CaM inhibitors did not
affect cell migration. These observations corroborate
organ culture studies in which TFP and W-7 inhibited
corneal epithelial movement in the rat but not in the
rabbit.13 CaM inhibitors reduced cell-to-substratum
adhesion in both species and this may be related to
the inhibition of stress fibers. In organ cultures of
corneas, cell-to-substratum adhesion is further reduced
by the inhibition of hemidesmosomes by CaM inhibitors.34
Corneal epithelial wound healing consists of a
premitotic movement of the epithelium to cover a
Table 1. Mean length of epithelial migration
at 48 hr on glass and collagen substrata
Glass
Collagen
Rat
Rabbit
832 (82)*
988(146)
805 (63)
1051 (232)
* (Standard deviation).
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Fig, 8. Distribution of F-actin at the leading edge in rat following
24 hr of treatment with CaM inhibitors: A, (TFP 20 nM) A
lamellopodium shows almost total loss of stress fibers. F-actin is
concentrated in the cortical cytoplasm. A diffuse, cytoplasmic
staining for F-actin is also present. B, (TFP 20 pM) A different
area of a leading edge shows similar loss of stress fibers; F-actin,
once again, is concentrated in the cytoplasm (bar = 10 ^m). W-7
results not shown.
defect as quickly as possible, followed by a mitotic
phase to restore the cell population.3536 A combination
of individual cell migration, cell enlargement, and
alterations in cell shape may contribute to the initial
premitotic movement. 3.35-38 Perhaps in the rabbit, the
premitotic phase of epithelial wound healing is comprised primarily of cell enlargement35 and alterations
in cell shape to move the tissue in a sliding fashion.38
In sheet-sliding movements, originally described in
rabbit corneal epithelium, no specific cytoplasmic
constituents supporting cell movement, such as alterations in the arrangement or density of cytoplasmic
filaments, were observed.38 It is therefore plausible
that stress fibers may not play a significant role in
these alternative mechanisms. Aside from rabbit corneal epithelium, there are other motile cells, such as
neural crest cells, granulocytes, monocytes, and amebas, that do not form stress fibers during migration.30
Herman et al postulated that stress fibers per se may
not be essential for motility, but that these structures
Fig, 9. Scanning electron micrographs of the leading edge in rat. A, Drug-free control shows multiple cytoplasmic extensions of the cell
membrane (filopodia, lamellopodia, and microplicae). B, After treatment with TFP 20 nM, note the smooth surface morphology, the
detachment of the cell edges from the substratum, and the loss of all cytoplasmic extensions. Tiny cracks on the cell surface are artifacts
(bar = 1 Mm),
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1985
Fig. 9C. Scanning electron micrograph of the central region away
from the leading edge after treatment with TFP 20 MM shows
intact cell surface extensions (microvilli and microplicae) (bar = 1
fim). W-7 results not shown.
which are intimately associated with adhesion plaques,
have a primary role in anchoring the cell to the
substratum and not in generating the actual loco-
Fig. 10. F-actin distribution following treatment with cytochalasin
B {5 fig/m]). A, Rat. Stress fibers have disappeared and F-actin
appears as multiple, diffusely scattered, punctate spots throughout
the cytoplasm. B, Rabbit. Punctate foci of F-actin have mostly
replaced the continuous peripheral band of F-actin that used to
delineate the cortical cytoplasm. Some rounding of cells is present
in both rat and rabbit (bar = 10 ^m).
•.
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Vol. 26
motive force itself.30 It would follow that the extensive
stress fiber pattern at the leading edge of rat corneal
epithelium may signify that this region has the greatest
cell-substratum adhesion as DiPasquale31 suggested.
The region behind the leading edge, having fewer
stress fibers and consequently less cell-substratum
adhesion, would then be the zone of maximum
sliding movement with the leading edge functioning
as an anchor. The leading edge itself continuously
seeks new sites of adhesion by extending pseudopodia.
Stress fibers have been observed around "wounds"
made in confluent rat aortic cells in culture39 and in
corneal endothelium and lens epithelium of frogs and
rats in situ adjacent to a wound.19 A similar statement
does not apply to rabbit corneal epithelium which
exhibits no obvious stress fibers or focal contacts.
Our results suggest that corneal epithelial migration
in rats is CaM-dependent, while it is not in rabbit.
CaM inhibitors appear to block migration best in
cells that elaborate stress fibers during spreading. For
instance, in cultured HeLa, mouse 3T3, kangaroo
PtK2, and rat mammary carcinoma cells, which all
have abundant stress fibers during locomotion, TFP
(40-80 nM) arrested cell motility and produced
changes in actin distribution and cell shape40 that
were identical to our cultured rat corneal epithelium.
Furthermore, CaM inhibitors appeared to affect leading edge cells more than the trailing cells. CaM
inhibitors could retard epithelial cell migration either
by direct antagonism of actin polymerization, by
reducing stress fiber formation, by acting on CaM,
by preventing myosin activation, by altering the cell
membrane, by blocking cell metabolism, by reducing
cell-to-substratum adhesion at the leading edge, or
by a combination of these mechanisms. The identical
effects of two structurally distinct CaM inhibitors,
TFP and W-7, and the reversibility of these effects
strongly indicate that the observed results were specific
to CaM inhibition and not caused by toxicity or
nonspecific side effects. In the rabbit, the specific
step(s) blocked by CaM inhibitors appears to be not
critical or rate-limiting in the maintenance of cell
motility.
CB, an inhibitor of actin polymerization, severely
affected the organization and distribution of F-actin,
and consequently blocked cell migration in both rat
and rabbit corneal epithelium. This reinforces the
view that actin polymerization is a common prerequisite to cell motility in both species.
Clinically, several classes of commonly used topical
eye medications, including the local anesthetics and
the beta-adrenergic blockers, possess strong CaMinhibitory effects.41 Moreover, these medications have
been implicated in causing persistent, nonhealing
corneal epithelial defects.42"45 It is suggested that
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ACTIN AND CALMODULIN IN CORNEAL EPITHELIAL HEALING / Soong and Cinrron
epithelial sloughing and delayed wound closure may
be due in part to the disruption of epithelial cell
microfilaments,42 whose maintenance and assembly
is thought to be CaM-dependent.
Previous organ culture13 and present cell culture
studies have shown striking differences between corneal epithelial healing dynamics in rat and rabbit.
We do not yet have sufficient information to elucidate
the precise mechanisms responsible for the difference.
Time-lapse photography, antibody localization of
CaM, and microinjection of CaM, CaM inhibitors,
and labelled actin may provide further clues as to the
mechanisms. The results of our present tissue culture
study may not necessarily represent corneal epithelial
healing mechanisms in vivo and caution is urged in
attempts to closely correlate these two systems. Moreover, tissue culture requirements may be critically
different between rat and rabbit; this possibility may
be difficult to rule out.
11.
12.
13.
14.
15.
16.
17.
Key words: actin, calmodulin, cell migration, corneal epithelium, phallacidin
18.
Acknowledgments
19.
The authors thank H.C. Covington, for electron microscopy; S. Brennan, for photography; S. Vitelli (Smith, Kline,
and French Laboratories; Philadelphia, PA), for generously
providing TFP; Dr. I. Gipson, for helpful discussions; and
Dr. D. Musch, for statistical help.
20.
21.
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